Restricted availability of donor tissue limits access to corneal transplantation for millions of individuals worldwide. Efforts are under way to produce artificial keratoprostheses and bio-engineered corneas for transplantation, however, currently there is no ideal material to serve as stroma. Synthetic substrates do not integrate well into the ocular environment and artificial collagen gels lack adequate strength and transparency. The long term goals of the project are to develop prosthetic cornea assembled from living corneal cells. This initial phase tests the hypothesis that acellular non-human corneal stromal matrix will provide an effective scaffolding on which a corneal equivalent can be assembled invitro. The project will identify decellularization and extraction procedures for bovine stromal tissue that eliminates cellular debris, maximize tissue repopulation by keratocytes, and supports expression of keratocyte differentiated function. Three specific aims will test these properties of stromal matrix in increasingly complex biological environments: (1) Examine the influence of acellular stromal matrix prepared by various procedures in promotion of repopulation and differentiated function of keratocytes in vitro. Corneas will be decellularized by a combination of freezing, hypotonic lysis, enzyme digestion, and detergent extraction. Associated tissue antigens and growth factors will be removed by a variety of extraction procedures. These decellularized tissues will then be used as culture matrix for primary and passaged keratocytes. Expression of markers of keratocyte differentiated phenotype will be examined. (2) Assess the role of epithelial and endothelial cells in the determining the differentiated functions of [unreadable] keratocytes in acellular stromal matrix. Matrix populated with keratocytes will be assembled into a three dimensional corneal equivalent with primary epithelial and endothelial cells. Stromal deturgescence, epithelial differentiation and basal lamina formation will be examined in addition to keratocyte function. (3) Compare the re-cellularization, transparency, and tissue rejection of acellular stromal matrix preparations in interlamellar grafts in vivo in mice. Acellular matrix successful in previous procedures will be transplanted in a mouse interlamellar model. The transplants will be examined for tissue rejection, recellularization, and transparency. These three aims will establish the suitability of stromal matrix as a scaffold for prosthetic corneas and determine the most effective methods for its preparation. [unreadable] [unreadable]